Colorimetric determination of the activity of methyltransferase based on nicking enzyme amplification and the use of gold nanoparticles conjugated to graphene oxide.

A method is described for the colorimetric determination of the activity of CpG methyltransferase (M.SssI). It is based on (a) the crosslinking effect between dsDNA-modified gold nanoparticles (AuNPs) and graphene oxide (GO), and (b) an amplification reaction with the aid of a nicking enzyme. To avoid the aggregation of AuNPs (which would produce false signals), a hairpin DNA was connected to the AuNPs. Thus, the red color of the solution (measured at 530 nm) increases linearly with the activity of M.SssI from 0.2 to 60 U·mL-1, and the limit of detection is 67 U·mL-1. This is superior to some reported strategies. The method was successfully applied to analyze spiked serum samples. Conceivably, it represents a powerful tool for use in drug development and diagnosis. Graphical abstracts A method based on the conjugated cross-linking effect between dsDNA modified Au NPs and GO coupled with an amplification reaction of nicking enzyme has been developed for colorimetric detection of the activity of CpG methyltransferase (M.SssI).

Fluorometric determination of the activity of the biomarker terminal deoxynucleotidyl transferase via the enhancement of the fluorescence of silver nanoclusters by in-situ grown DNA tails.

The activity of terminal deoxynucleotidyl transferase (TdTase) is a biomarker for routine diagnosis of acute leukemia. A method has been developed for the determination of TdTase activity. It is based on the use of silver nanoclusters (AgNCs) whose yellow fluorescence is enhanced by an in-situ grown DNA tail of TdTase-polymerized and guanine-rich DNA at the 3' end of a hairpin DNA. The fluorescence, best measured at excitation/emission peaks of 530/585 nm, increases linearly in the 1 to 35 mU mL-1 TdTase activity range. The detection limit is 0.8 mU mL-1. The method is cost-efficient, selective and convenient. It integrates enhancement of the fluorescence of AgNCs and target recognition into a single process. Graphical abstract Schematic presentation of a method for determination of TdTase activity. It is based on AgNCs fluorescence enhanced by in-situ grown TdTase-polymerized G-rich DNA tail. The method integrates AgNCs fluorescence enhancement and the target recognition into a single process.

Two new steroids from sclerotia of the fungus Omphalia lapidescens.

Two new steroids, leiwansterols A and B, along with three known ones (3-5), were isolated from the sclerotia of the fungus Omphalia lapidescen. Their structures were elucidated on the basis of spectral data. The isolated compounds showed weak anti-tobacco mosaic virus activity.

Sex differences in a corticosterone-induced depression model in mice: Behavioral, neurochemical, and molecular insights.

Depression is characterized by a significant sex disparity, with higher rates observed in women compared to men. This study aimed to investigate the impact of sex on depressive behaviors and explore the underlying mechanisms using a corticosterone (CORT)-induced depression model in mice. Behavioral tests, Nissl staining, UPLC-MS/MS, and Western blot analysis were performed to assess behavioral changes, as well as neuronal alterations, neurotransmitter levels, and protein expressions in the hippocampus. The mice in the model group exhibited sex-specific anxiety- and depression-like behaviors. Nissl staining revealed structural abnormalities in the CA3 region of the hippocampus in females. Neurotransmitter analysis indicated decreased serotonin and norepinephrine levels in both sexes, while glutamate levels were elevated in females. Furthermore, female mice demonstrated elevated serum CORT levels. Western blot analysis revealed sex-specific alterations in specific protein expression. Female mice exhibited downregulated glucocorticoid receptor and brain-derived neurotrophic factor expression, whereas male mice showed minimal changes. Additionally, female mice displayed reduced phosphorylated AKT, phosphorylated PI3K, and phosphorylated mTOR levels. These findings enhance our understanding of sex-specific differences in the CORT-induced depression model and provide insights into the underlying mechanisms of depression. This research emphasizes sex in depression studies and supports tailored interventions.

[Neurotrophic effects of senegenin].

OBJECTIVE: To study the neurotrophic effects of senegenin on the expression of MAP2 mRNA and BDNF mRNA in cultured cerebral cortical neurons. METHODS: The newborn rat cerebral cortex neurons were cultured in vitro. LDH assay was used to investigate the effect of senegenin on the neuronal viability and reverse transcription polymerase chain reaction (RT-PCR) was carried out to determine the expression level of MAP2 mRNA and BDNF mRNA. RESULTS: LDH assay showed that senegenin at the concentration of 0. 5 micromol/L,1 micromol/L and 2 micromol/L could obviously enhance the survival of cells and the survival rates were in dose-dependent manner to some extent. Moreover, the low, medium and high concentrations of senegenin significantly increased the expression of MAP2 mRNA and BDNF mRNA. CONCLUSION: The study suggests that suitable dose of senegenin can increase the expression of MAP2 mRNA and BDNF mRNA in cultured cerebral cortical neurons, and its mechanism needs further study.

[Neurotrophic effects of protocatechuic acid on neurite outgrowth and survival in cultured cerebral cortical neurons of newborn rat].

OBJECTIVE: To study the neurotrophic effects of protocatechuic acid on neurite outgrowth and survival in cultured cerebral cortical neurons. METHODS: The newborn rat cerebral cortex neurons were cultured in vitro. The convert phase microscope was used to count the survival neurons with neurites and measure the average length of neurites. MTT and LDH assay were carried out to investigate the effect of PCA on the neuronal viability. RESULTS: Compared with control group, different concentration of PCA could increase the number of survival neurons with neurites and the average length of neurites. MTT and LDH assay showed that PCA promoted neuron survival in a dose-dependent manner. CONCLUSION: PCA can enhance the survival of rat cortical neurons with neurites and promote the neurite outgrowth of rat cortical neurons.

Label-free fluorescence strategy for methyltransferase activity assay based on poly-thymine copper nanoclusters engineered by terminal deoxynucleotidyl transferase.

The assay of detecting DNA methyltransferase activity has been identified as one of the central challenges in cancer diagnostics as DNA methylation is closely related to the diagnosis and treatment of tumors. In this study, a label-free fluorescence probe based on poly-thymine copper nanoclusters engineered by terminal deoxynucleotidyl transferase is proposed for methyltransferase activity assay. Taking advantage of the efficient polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase and the copper nanoclusters with large Stokes shift instead of labeling fluorescent dyes, the strategy exhibits a broader linear scope from 1 to 300 U mL-1 with a detection limit of 0.176 U mL-1. The economical method is specificity for M.SssI and also provides a convenient and high-throughput platform for screening the DNA methylation inhibitors, which displays a great potential for the practical applications of the drug development and clinical cancer diagnosis in the future.

Label-free and enzyme-free one-step rapid colorimetric detection of DNA methylation based on unmodified gold nanoparticles.

DNA methylation has been identified as one of the important causes of tumorigenesis, so it is important to develop some advanced methods for detecting and quantifying DNA methylation. In this study, a label-free and enzyme-free one-step rapid colorimetric detection of DNA methylation based on unmodified Au nanoparticles(Au NPs)has been proposed. This method can quickly, efficiently, economically and easily colorimetric detect methylated DNA only by the color change of unmodified Au NPs solution without the covalent modification of Au NPs in advance or complicated instruments for implementation with practical limitations or expensive biological enzymes or traditional organic dyes during the reaction. The strategy employed the difference in electrostatic attraction of single-stranded DNA and double-stranded DNA against salt-induced aggregation of Au NPs. The method has a DNA methylated detection limit of 8.47 nM and it is distinctly visible to detect methylated DNA with the naked eye as low as 20 nM. Furthermore, the strategy has an ability to detect methylated DNA in the presence of abundant unmethylated DNA with the detection limit of 0.13% and as low as 1% methylated DNA can be distinguished in heterogeneous samples with the naked eye. Also, the stratagem provides a convenient and rapid platform for methylated DNA detection of human serum samples in one step, which displays a huge potential for clinical diagnosis and treatment of oncological diseases.

Prevalence of the methylenetetrahydrofolate reductase 677C>T polymorphism in the pregnant women of Yunnan Province, China.

Mutations in the methylenetetrahydrofolate reductase (MTHFR) gene can result in a reduced ability to utilize folic acid. The MTHFR 677C>T polymorphism in particular has been linked to both birth defects and pregnancy-associated diseases. This study aimed to evaluate the prevalence of the MTHFR 677C>T mutation among pregnant women in Yunnan Province so as to collect baseline data that may be utilized to guide folic acid supplementation efforts and to support related disease prevention programs. We retrospectively reviewed 3387 pregnant women from Yunnan Province. The MTHFR 677C>T polymorphism was identified using polymerase chain reaction (PCR) and DNA sequencing. In total, 1350 (39.9%) subjects were homozygous for the C allele (CC), 1540 (45.4%) subjects were heterozygous (CT), and 497 (14.7%) subjects were homozygous for the T allele (TT). The MTHFR 677C>T polymorphism was found to be present within the studied population, with ∼60% of these patients being either heterozygous or homozygous for the mutant allele and with an overall T allele frequency of 0.37. The frequency of the T allele was significantly higher among pregnant women with complications relative to women with healthy pregnancies, particularly among women <30 years old. As such, the maternal MTHFR 677C>T polymorphism may be a genetic risk factor associated with pregnancy complications and may help identify pregnant women at a high risk of such complications.

Fluorescence-based Polymerase Amplification for the Sensitive Detection of DNA Methyltransferase Activity.

DNA methyltransferase (MTase) is related to transcriptional repressor activity in biological functions. It is an essential for cancer diagnosis and therapeutics to detect DNA MTase activity sensitively. Here, a fluorescent system based on polymerase amplification has been developed to detect DNA adenine MTase (Dam) activity sensitively. The amplification is triggered by the probe DNA regions a, which are the primes of a polymerase-induced replicated reaction. They come from methylation and a digestion reaction of DNA S1-S1, including a 5'-GATC-3' sequence recognized by Dam MTase and methylation sensitive restriction endonuclease Dpn I. The intensities of fluorescence are dependent on the Dam MTase activity. The method shows fine sensitivity with a detection limit of 3.2 × 10-4 U mL-1 and specificity for Dam MTase. In human serum samples, the method has been successfully applied, and it has also been used to screen the inhibitors, which means that the developed method can be a powerful and potential tool for drug development and clinical diagnosis in the future.

PI3K/Akt signaling pathway is involved in the neurotrophic effect of senegenin.

Neurodegenerative diseases are frequently associated with the loss of synapses and neurons. Senegenin, extracted from the Chinese herb Polygala tenuifolia Willd, was previously found to promote neurite outgrowth and neuronal survival in primary cultured rat cortical neurons. The aim of the present study was to investigate the underlying mechanisms of senegenin-induced neurotrophic effects on rat cortical neurons. Primary cortical rat neurons were treated with various pharmacological antagonists and with or without senegenin, and subjected to MTT and western blot analysis to explore the effects of senegenin on cell survival as well as the activation of signaling pathways. Neurite outgrowth and neuronal survival induced by senegenin were significantly inhibited by A2A receptor antagonist ZM241385 and specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002, but not by tropomyosin receptor kinase A receptor inhibitor K252a, mitogen-activated protein kinase kinase inhibitor PD98059 or protein kinase C inhibitor GÖ6976. Furthermore, senegenin enhanced the phosphorylation of Akt, which was blocked by LY294002. The present study revealed that the PI3K/Akt signaling pathway may be involved in the neurotrophic effects of senegenin.